The overall long-term goal of this project continues to be directed at understanding the involvement of zinc binding proteins and those proteins that interact with zinc for the absorption, metabolism, and biological function of dietary zinc. The project aims are interrelated. Aim 1. Functional Significance of Metallothionein (MT) Expression in Human and Murine Cells and Tissues. Using quantitative PCR (Q-PCR), assays for hMT and specific zinc transporter genes of both the SLC30 and SLC39 families will be used in controlled zinc depletion and zinc supplementation studies with human subjects to evaluate the zinc responsiveness of these genes in monocytes and T cells. The influence of murine MT, as regulated by dietary zinc, will be evaluated as a factor in nitrosative stress. These experiments will use MT null and inducible nitric oxide (NO) synthase null genotypes and focus on isolated liver parenchymal cells in culture and targets of NO, viz., intestinal cells. Aim II. Characterization of Zinc Responsive Genes. These studies will focus on THP1 cells as a model for human mononuclear cells using chelator-induced zinc depletion and supplementation. Comparisons will be made between zinc-responsive genes [unreadable] LPS activation. Similar studies will compare zinc-responsive genes of thymocytes [unreadable] LPS from zinc-deficient and zinc-supplemented mice. Analysis will be by microarrays, with confirmation by proteomics. Responsiveness of the MTF1 and MTF2 transcription factors to dietary zinc will be compared. Aim III. Murine Zinc Transporters and Their Response to Dietary Zinc Intake and Physiologic Mediators. The IL-6 responsive transporter Zip14 will be further characterized with transfected cells and for a role in inflammatory hypozincemia. Expression of zinc transporter genes of the reticuloendothelial system during the acute phase response will be evaluated. The zinc processing steps the pancreatic acinar cells and Paneth cells use for endogenous zinc release will receive close attention. Extensive use will be made of transporter localization using immunohistochemistry and immunocytochemistry and zinc fluorophores as visualized by fluorescence microscopy.